Targeted vectors for cancer immunotherapy

ABSTRACT

This invention provides compositions and methods for treating cancer. More specifically this invention is directed to a targeted retroviral vector comprising a cytokine gene that can be administered either alone or in combination with a targeted retroviral vector comprising a cytocidal gene for treating cancer in a subject. Also provided are a kit or drug delivery system comprising the compositions for use in the methods described.

1. RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) ofprovisional application serial number 60/250,185 filed Nov. 29, 2000,the disclosure of which is hereby incorporated by reference in itsentirety.

2. FIELD OF THE INVENTION

[0002] This invention is in the field of oncology, more specificallythis invention relates to targeted injectable vectors, such as targetedretroviral particles, for use in cancer immunotherapy.

3. BACKGROUND OF THE INVENTION

[0003] Immune modulation in conjunction with tumor antigen presentationis a promising approach for optimizing the efficacy of cancer genetherapy protocols for metastatic cancer or minimal residual disease. Intumor vaccine strategies, cytokines such as granulocyte-macrophagecolony-stimulating factor (GM-CSF) are employed to recruitantigen-presenting cells, including dendritic cells and macrophages,which result in the activation of cytotoxic T lymphocytes (CTL) againstproteins expressed by cancer cells (Warren and Weiner, 2000; Kim et al.,2000). GM-CSF induces activation, proliferation, and differentiation ofa variety of immunologically active cell populations, therebyfacilitating the development of both humoral and cellular-mediatedimmunity (Warren and Weiner, 2000). One promising vaccine approachinvolves the insertion of the GM-CSF gene into autologous cancer cellsthat are then used for immunization (Jaffee, 1999; Suh et al., 1999).These genetically engineered tumor cells produce the GM-CSF protein inthe local environment of tumor cells, thereby activating the patients Tcells, which then function to eradicate the cancer at metastatic sites.Whether delivered as genetically engineered tumor cells or as thesoluble GM-CSF protein, the cytokine must be present in the same site asthe vaccine component (Mellstedt et al., 1999). Indeed, theestablishment of specific and long lasting antitumor immunity followingvaccination with GM-CSF tumor cells requires the simultaneous presenceof GM-CSF and tumor antigens at the vaccination site (Nagai et el.,1998). However, in spite of the therapeutic potential demonstrated inanimal models and early-phase clinical trials, the clinical developmentof these protocols has been limited by difficulties relating to theestablishment of autologous tumor cell cultures (Fong et al., 2000) andthe performance of individualized gene transfer procedures ex vivo(Borrello et al., 1999). Alternatively, local delivery of GM-CSF bydirect intratumoral injection, as well as paracrine secretion bygenetically engineered cells, has been shown to be more effective inupregulating lymph node sensitization when compared to systemicadministration (Kurane et al., 1997).

[0004] A novel cancer immunotherapy approach for metastatic cancer wouldexploit the potential of systemically administered matrix-targetedretroviral vectors, infused sequentially intravenously, to efficientlydeliver both a cytokine gene and/or a cytocidal construct to tumor cellsand associated tumor vasculature. The efficacy of matrix-targeted genedelivery has been demonstrated in models of liver metastasis (Gordon etal., 2000a) and subcutaneous human cancer xenografts in nude mice(Gordon et al., 2000b). The present invention provides targetedretroviral particles, for systemic administration, carrying one or morecytokine genes that provide high level efficiency of cytokine genedelivery into a tumor and recruitment of host mononuclear cells (tumorinfiltrating lymphocytes) into the tumor.

4. SUMMARY OF THE INVENTION

[0005] This invention relates in general to compositions and methods foruse in cancer immunotherapy. More particularly this invention isdirected to targeted injectable vectors for use in cancer immunotherapy.

[0006] It is an object of this invention to provide a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector and a cytokine gene.

[0007] It is an object of this invention to provide a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector to an extracellular matrix component or tumorvasculature and a cytokine gene.

[0008] It is a further object of this invention to provide a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle to an extracellular matrix componentcomprising a binding region which binds to an extracellular matrixcomponent and a cytokine gene (e.g., granulocyte-macrophagecolony-stimulating factor (GM-CSF)).

[0009] It is yet another object of this invention to provide a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle (e.g., targeting an extracellular matrixcomponent or tumor vasculature ) and a cytokine gene (e.g., GM-CSF) anda targeted retroviral particle comprising a modified viral surfaceprotein for targeting the vector particle (e.g., targeting anextracellular matrix component or tumor vasculature) and a cytocidalgene (e.g., tumor suppressor genes).

[0010] It is another object of this invention to provide a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector (e.g., targeting an extracellular matrix componentor tumor vasculature) and a cytokine gene (e.g., GM-CSF) and/or atargeted retroviral particle comprising a modified viral surface proteinfor targeting the vector particle (e.g., targeting an extracellularmatrix component or tumor vasculature ) and a cytocidal gene (e.g.,tumor suppressor genes) to be administered intravenously orintra-arterially.

[0011] Yet another object of this invention is a method of treatingcancer in a subject comprising administering a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector and a cytokine gene either alone or in conjunction with atargeted retroviral particle comprising a modified viral surface proteinfor targeting the vector particle and a cytocidal gene.

[0012] It is a further object of this invention to provide a method oftreating primary tumors or tumors located in surgically inaccessiblesites in a subject comprising administering a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector and a cytokine gene either alone or in conjunction with atargeted retroviral particle comprising a modified viral surface proteinfor targeting the vector particle and a cytocidal gene.

[0013] It is yet another object of this invention to provide methods ofadministering targeted injectable vectors, such as targeted retroviralparticles to achieve high level efficiency of cytokine gene deliveryinto distant tumor sites resulting in secretion of GM-CSF by transducedtumor cells within the solid tumor and/or recruitment of host immunecells (e.g., mononuclear cells), such as tumor infiltrating lymphocytes(TIL) into the tumor.

[0014] It is a further object of this invention to provide a kit or drugdelivery system comprising the compositions for use in the methodsdescribed herein.

5. BRIEF DESCRIPTION OF FIGURES

[0015]FIG. 1 shows a schematic diagram of the molecular engineering ofthe GM-CSF retroviral expression vector. The retroviral expressionvector (pREX II) was created by engineering a multiple cloning site(MSC) into the G1XSvNa vector (Genetic Therapy, Inc.) to produce G1(MCS)SvNa (A), which is then subjected to Kpn I digestion followed byfusion of the Kpn I fragment (C) with the linearized pRV109 vector (B).The resulting pREX II retroviral expression vector (D) is driven by ahybrid CMV/MSV/MLV promoter at the 5′ LTR and a standard MLV LTR at the3′ end. The 0.44 kb cDNA encoding human granulocyte macrophage colonystimulating factor (GM-CSF), was cloned into the unique Not 1 (5,) andXho 1 (3,) cloning sites of the pREX II vector (E).

[0016]FIG. 2 shows the immunologic detection of human GM-CSF proteinsecreted by transduced NIH3T3 cells and transfected 293T cells. (A) Thedarkened ELISA wells indicate immunoreactive human GM-CSF secreted bytransduced NIH3T3 and transfected 293T cells, as detected by apolyclonal goat anti-human GM-CSF IgG (R&D Systems, Inc.). (B) Serialdilutions of supernatant collected from transduced NIH3T3 andtransfected 293T cell cultures were used to measure immunoreactive humanGM-CSF protein (shown as decreasing immunoreactivity with eachdilution), against a purified rhGM-CSF standard.

[0017]FIG. 3 shows high level transduction of the tumor nodule byintravenous injection of the matrix-targeted retroviral vector bearing anuclear targeted β-galactosidase gene. (A: X 400) A negative stainingcontrol tumor nodule from Mx-null vector-treated animal. (B: X400) Atumor nodule from a Mx-nBg vector-treated animal. β-galactosidaseexpressing tumor cells (closed arrows) and tumor endothelial cells (openarrows) are shown as reddish-brown nuclear stained cells, counterstainedwith methyl green.

[0018]FIG. 4 is a histologic section of a tumor nodule expressingimmunoreactive human GM-CSF from nude mice treated with intravenousinjections of the matrix-targeted GM-CSF retroviral vector. (A: X 40) Anegative staining control tumor nodule from Mx-null vector-treatedmouse. (B: X40) A tumor nodule from a Mx-Gm-CSF vector-treated animal.Human GM-CSF expressing tumor cells are shown as reddish-brown nuclearstained cells, counterstained with methyl green. (C: X200) Boxed area inB with human GM-CSF expressing tumor cells are indicated by arrows. (D:X40) Tumor nodule in B as negative control with no primary antibody.

[0019]FIG. 5 shows extensive infiltration of the tumor nodule by hostmononuclear cells (tumor infiltrating lymphocytes) in mice treated withintravenous injections of the matrix-targeted GM-CSF retroviral vector.(A: 40X) H&E stained tissue section of tumor nodule from a Mx-nullvector-treated mouse. (B: X40) H&E stained tissue section of tumornodule from a Mx-GM-CSF vector-treated animal. (C: X1 00) Highermagnification of A (boxed area) showing a pleomorphic population oftumor cells, tumor stromal and endothelial cells with minimalmononuclear cell infiltration. (D: X100) Higher magnification of Bshowing extensive infiltration of host mononuclear cells.

6. DETAILED DESCRIPTION

[0020] Throughout this disclosure, various publications, patents andpublished patent specifications are referenced by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications are hereby incorporated by reference into thepresent disclosure to more fully describe the state of the art to whichthis invention pertains.

[0021] Definitions

[0022] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of immunology, molecularbiology, microbiology, cell biology and recombinant DNA. These methodsare described in the following publications. See, e.g., Sambrook, et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) edition (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.); “PCR: A PRACTICALAPPROACH” (M. MacPherson, et al., IRL Press at Oxford University Press(1991)); PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames andG. R. Taylor eds. (1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow andLane, eds. (1988)); and ANIMAL CELL CULTURE (R. I. Freshney, ed.(1987)).

[0023] As used in the specification and claims, the singular form “a”,“an” and “the” include plural references unless the context clearlydictates otherwise. For example, the term “a targeted retroviralparticle” includes a plurality of particles, including mixtures thereof.

[0024] The term “cancer” includes a myriad of diseases generallycharacterized by inappropriate cellular proliferation, abnormal orexcessive cellular proliferation. Examples of cancer include but are notlimited to, breast cancer, colon cancer, prostate cancer, pancreaticcancer, melanoma, lung cancer, ovarian cancer, kidney cancer, braincancer, or sarcomas. Such cancers may be caused by, chromosomalabnormalities, degenerative growth and developmental disorders,mitogenic agents, ultraviolet radiation (UV), viral infections,inappropriate tissue expression of a gene, alterations in expression ofa gene, or carcinogenic agents.

[0025] The term “treatment” includes, but is not limited to, inhibitionor reduction of proliferation of cancer cells, destruction of cancercells, prevention of proliferation of cancer cells or prevention ofinitiation of malignant cells or arrest or reversal of the progressionof transformed premalignant cells to malignant disease or ameleriationof the disease.

[0026] The term “subject” refers to any animal, preferably a mammal suchas a human vetinary uses are also intended to be encompassed by thisinvention.

[0027] The present invention provides, in general, compositions andmethods for use in cancer immunotherapy. More particularly, thisinvention is directed to targeted injectable vectors, such as targetedretroviral particles, for use in cancer immunotherapy wherein thetargeted retroviral particle comprises a modified viral surface proteinfor targeting the vector particle and is capable of expressing acytokine gene. These compositions and methods are based on anobservation by the inventors that a targeted retroviralparticlesexpressing a cytokine gene can be administered systemically andachieve (i) high level efficiency of cytokine gene delivery into solidtumors, (ii) secretion of GM-CSF by tumor cells within the solid tumor,and (iii) recruitment of host mononuclear cells (tumor infiltratinglymphocytes, TIL) into the GM-CSF secreting tumor nodules foreradication of primary, metastatic, inaccessible or minimal residualdisease.

[0028] In one embodiment the invention provides a targeted injectablevector in the form of a targeted retroviral particle comprising acytokine gene. The retroviral particle is targeted to cancer cells ortumors by modifying the viral surface protein to express a targetingpolypeptide. Preferably, the targeting polypeptide recognizes motifsassociated with cancer or tumor growth. Examples of motifs forrecognition by the targeting polypeptide include, but are not limitedto, extra cellular matrix (ECM) or tumor vasculature (see e.g.,WO/01/07059 and WO/01/31036, hereby incorporated by reference). By wayof example, the targeting polypeptide could recognize the extra cellularmatrix (ECM), endothelial cells or stromal cells exposed during tumorgrowth, angiogenesis or metastasis. Examples of collagen binding motifsthat may be used to target the ECM include, but are not limited to, thecollagen binding motif derived from the von Willebrand coagulationfactor (Takigi, J., et al (1992) Biochemistry 32:8530 and WO/01/07059).

[0029] The targeted retroviral particle can be generated by conventionalmethods. By way of example, a three plasmid co-transfection system maybe used to construct the retroviral particle. Generally, in such asystem, one plasmid comprises the gag-pol genes, a second plasmidcomprises the chimeric or hybrid surface proteins for targeting theretroviral particle, and the third plasmid comprises an expressionvector containing the cytokine gene (e.g., Examples 1 and 2; Miller(1990) Human Gene Therapy Vol. 1, U.S. Pat. No. 5,952,225; Soneoka et al(1995) Nucl. Acid Research 23:628;WO/01/07059 and WO/01/31036, herebyincorporated by reference).

[0030] Expression vectors suitable for use in expressing the cytokinegene may comprise at least one expression control element operablylinked to the nucleic acid sequence encoding the cytokine gene.Expression control elements may be inserted in the vector to control andregulate the expression of the cytokine nucleic acid sequence. Examplesof expression control elements include, but are not limited to, lacsystem, operator and promoter regions of phase λ, yeast promoters, andpromoters derived from polyoma, adenovirus, retroviruses, or SV40. Thevector may further comprise additional operational elements including,but not limited to, leader sequences, termination codons,polyadenylation signals, and any other sequences necessary or preferredfor the appropriate transcription and/or translation of the cytokinenucleic acid sequence. It will be understood by one skilled in the artthat the correct combination of required or preferred expression controlelements will depend on the gene to be expressed, target tissue and thelike. It will be further understood by one skilled in the art that suchvectors are constructed using conventional methodology (See e.g.Sambrook et al., (eds.) (1989) “Molecular Cloning, A laboratory Manual”Cold Spring Harbor Press, Plainview, N.Y.; Ausubel et al., (eds.) (1987)“Current Protocols in Molecular Biology” John Wiley and Sons, New York,N.Y.; WO/01/31036, hereby incorporated by reference) or are commerciallyavailable.

[0031] In a preferred embodiment, the targeted retroviral particles areviral vectors. Examples of viral expression vectors that may be usedinclude, but are not limited to, retroviral vectors, such as lentivirus,vaccinia virus vectors, adenovirus vectors, herpes virus vector, or fowlpox virus vector. In a preferred embodiment, the cytokine gene isincorporated into a retroviral expression vector and packaged into theretroviral particle (see e.g., W/01/07059 and WO/01/31036, herebyincorporated by reference).

[0032] Cytokines modulate the immune system. The cytokine geneincorporated into the expression vector may be any cytokine gene,preferably a cytokine which enhances or stimulates the humoral orcellular immune response. The polynucleotide encoding the gene may beDNA or RNA. The targeted retroviral vector may comprise a polynucleotideencoding the entire cytokine protein or a polynucleotide encoding aportion of the protein necessary for the biological activity of thecytokine. Cytokines include but are not limited to, interleukins,lymphokines, monokines, interferons, colony stimulating factors andchemokines. Examples of specific cytokine that may be used include, butare not limited to, IL-1, TNF, IL-2, IFN-γ, IL-4, IL-7 and GM-CSF.Preferred cytokines are GM-CSF and IL-2. The targeted retroviral vectorsmay comprise all or part of one or more cytokine genes.

[0033] In another embodiment this invention also provides a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector (e.g., targeting an extracellular matrix componentor tumor vasculature) and a cytocidal gene (e.g., tumor suppressorgenes). These targeted retroviral particles may be generated asdescribed herein above, (see, e.g., WO/01/64870) The cytocidal genemaybe any gene which inhibits, destroys or prevents cancer cell growthor induces apoptosis in cancer cells. Examples of cytocidal genesincludes, but is not limited to, tumor suppressor genes (e.g., p53, RB),thymidine kinases (e.g., HSV thymidine kinase, CMV thymidine kinase) ormutated cyclin G1 genes. The vector may comprise a polynucleotideencoding for an entire cytocidal gene or a portion of a polynucleotideencoding a portion of the cytocidal protein sufficient to exert itsbiological activity. By way of example, the cytocidal gene may be adominant negative mutation of the cyclin G1 protein (e.g., WO/01/64870).The targeted retroviral particle comprising a modified viral surfaceprotein for targeting the vector and a cytocidal gene may be used aloneor in conjunction with the targeted retroviral particle comprising amodified viral surface protein for targeting the vector and a cytokinegene. Alternatively, both the cytokine and cytocidal gene may becontained within the same targeted retroviral vector.

[0034] In an alternative embodiment, the targeted injectable vector maybe in the form of nonviral vectors, such as cationic liposomesexpressing a cytokine gene or cytocidal gene.

[0035] Another embodiment of this invention relates to methods oftreating cancer with immunotherapy by administering the targetedretroviral particles described herein to a subject. The term treatmentas used herein is intended to include, but is not limited to,administration of the targeted retroviral particles prior to anyevidence of disease (e.g., subjects at risk of occurrence of the diseaseor at risk of recurrence) or to mediate regression of the disease in asubject. The quantity of targeted viral particle comprising a cytokinegene to be administered is based on the titer of virus particles. By wayof example, a range of particles to be administered is about 10⁹ toabout 10¹² colony forming units (cfu). After administration, theefficacy of the treatment can be assessed by cytokine production bytransduced tumors, recruitment into the tumor site of immune cells(e.g., TIL) or antibodies that recognize the tumor antigen, and/or bytumor regression. One skilled in the art would know the conventionalmethods to assess the aforementioned parameters.

[0036] The targeted retroviral particle comprising the cytokine gene maybe administered alone or in conjunction with other therapeutictreatments or active agents. For example, the targeted retroviralparticle comprising a cytokine gene may be administered with thetargeted retroviral particle comprising a cytocidal gene. The quantityof the targeted retroviral particle comprising a cytocidal gene to beadministered is based on the titer of the virus particles as describedherein above. By way of example, if the targeted retroviral particlecomprising a cytokine gene is administered in conjunction with atargeted retroviral particle comprising a cytocidal gene the titer ofthe retroviral particle for each vector may be lower than if each vectoris used alone. The targeted retroviral particle comprising the cytokinegene may be administered concurrently or separately from the targetedretroviral particle comprising the cytocidal gene.

[0037] The methods of the subject invention also relate to methods oftreating cancer by administering a targeted retroviral particle (e.g.,the targeted retroviral vector expressing a cytokine either alone or inconjunction with the targeted retroviral vector expressing a cytocidalgene) with one or more other active agents. Examples of other activeagents that may be used include, but are not limited to,chemotherapeutic agents, anti-inflammatory agents, protease inhibitors,such as HIV protease inhibitors, nucleoside analogs, such as AZT. Theone or more active agents may be administered concurrently or separately(e.g., before administration of the targeted retroviral particle orafter administration of the targeted retroviral particle) with the oneor more active agents. One of skill in the art will appreciate that thetargeted retroviral particle may be administered either by the sameroute as the one or more agents (e.g., the targeted retroviral vectorand the agent are both administered intravenously) or by differentroutes (e.g., the targeted retroviral vector is administeredintravenously and the one or more agents are administered orally).

[0038] An effective amount or therapeutically effective of the targetedretroviral particles to be administered to a subject in need oftreatment may be determined in a variety of ways. By way of example, theamount may be based on viral titer or efficacy in an animal model.Alternatively the dosing regimes used in clinical trials may be used asgeneral guidelines. The daily dose may be administered in a single doseor in portions at various hours of the day. Initially, a higher dosagemay be required and may be reduced over time when the optimal initialresponse is obtained. By way of example, treatment may be continuous fordays, weeks, or years, or may be at intervals with intervening restperiods. The dosage may be modified in accordance with other treatmentsthe individual may be receiving. However, the method of treatment is inno way limited to a particular concentration or range of the targetedretroviral particle and may be varied for each individual being treatedand for each derivative used.

[0039] One of skill in the art will appreciate that individualization ofdosage may be required to achieve the maximum effect for a givenindividual. It is further understood by one skilled in the art that thedosage administered to a individual being treated may vary depending onthe individuals age, severity or stage of the disease and response tothe course of treatment. One skilled in the art will know the clinicalparameters to evaluate to determine proper dosage for the individualbeing treated by the methods described herein. Clinical parameters thatmay be assessed for determining dosage include, but are not limited to,tumor size, alteration in the level of tumor markers used in clinicaltesting for particular malignancies. Based on such parameters thetreating physician will determine the therapeutically effective amountto be used for a given individual. Such therapies may be administered asoften as necessary and for the period of time judged necessary by thetreating physician.

[0040] While it is possible for the targeted retroviral particle to beadministered in a pure or substantially pure form, it is preferable topresent it as a pharmaceutical composition, formulation or preparation.

[0041] Pharmaceutical compositions comprising the targeted retroviralparticles to be used in the methods described herein may be formulatedand administered by methods well known in the art (Remington'sPharmaceutical Sciences, 20th Edition, Lippincott, William & WilkinsBaltimore, Md.). For example, the compositions of the present inventionmay comprise an effective amount of the targeted retroviral particlesand a pharmaceutically and/or physiologically acceptable carrier. Thecharacteristics of the carrier will depend on the route ofadministration. Such a composition may contain, in addition to thetargeted retroviral particles, diluents, filters, salts, buffers,stabilizers, solubilizers and other materials well known in the art.

[0042] The compositions of the may be formulated for various routes ofadministration by methods well known in the art, including, but notlimited to parenteral administration,( e.g., for injection via theintravenous, intramuscular, sub-cutaneous, intratumoral orintraperitoneal routes), oral administration or topical administration.In a preferred embodiment, the targeted retroviral particles areadministered intravenously or intra-arterially. Upon formulation, thecompositions are administered in a manner compatible with the dosageformulation. By way of example, the administration may vary from severaltimes a day to less frequent administrations, such as once a day orevery other day for only a few days such as with a rest period ofvarying lengths, such as a week. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Veterinaryuses are also intended to be encompassed by this invention.

[0043] It is a further object of this invention to provide a kit or drugdelivery system comprising the compositions for use in the methodsdescribed herein. All the essential materials and reagents required foradministration of the targeted retroviral particle may be assembled in akit (e.g., packaging cell construct or cell line, cytokine expressionvector). The components of the kit may be provided in a variety offormulations as described above. The one or more targeted retroviralparticle may be formulated with one or more agents (e.g., achemotherapeutic agent) into a single pharmaceutically acceptablecomposition or separate pharmaceutically acceptable compositions.

[0044] The components of these kits or drug delivery systems may also beprovided in dried or lyophilized forms. When reagents or components areprovided as a dried form, reconstitution generally is by the addition ofa suitable solvent, which may also be provided in another containermeans. The kits of the invention may also comprise instructionsregarding the dosage and or administration information for the targetedretroviral particle. The kits or drug delivery systems of the presentinvention also will typically include a means for containing the vialsin close confinement for commercial sale such as, e.g., injection orblow-molded plastic containers into which the desired vials areretained. Irrespective of the number or type of containers, the kits mayalso comprise, or be packaged with, an instrument for assisting with theinjection/administration or placement of the ultimate complexcomposition within the body of a subject. Such an instrument may be anapplicator, inhalant, syringe, pipette, forceps, measured spoon, eyedropper or any such medically approved delivery vehicle.

[0045] The following examples illustrate various aspects of theinvention, but in no way are intended to limit the scope thereof.

7. EXAMPLES Example 1 Construction of the GM-CSF Retroviral ExpressionVector

[0046] The retroviral expression vector (pREX 11) was created byengineering a multiple cloning site (MSC) into the G 1 XSvNa vector(Genetic Therapy, Inc.) to produce G 1 (MCS)SvNa (FIG. 1A), which isthen subjected to Kpn I digestion followed by fusion of the Kpn Ifragment (FIG. 1C) with the linearized pRV 109 vector (FIG. 1B). Theresulting pREX II retroviral expression vector (FIG. 1D) is driven by ahybrid CMV/MSV/MLV promoter at the 5′ LTR and a standard MLV LTR at the3′ end. Bearing the strong CMV promoter and an SV40 ori, this plasmid issuitable for high titer vector production in 293T cells prepared bytransient transfection protocols. (Soneoka et al., 1995). The 0.44 kbcDNA encoding human granulocyte macrophage colony stimulating factor(GM-CSF), GenBank accession number NM 000758, flanked by PCR-derivedrestriction sites was cloned into the unique Not 1 (5′) and Xho 1 (3′)cloning sites of the pREX II vector (E).

Example 2 Production of Matrix-targeted Retroviral Vectors Bearing aHuman GM-CSF Construct

[0047] High titer vectors were generated utilizing a transient threeplasmid co-transfection system (Soneoka et al., 1995) in which thepackaging components gag-pol, a chimeric MLV-based env bearing a vonWillebrand factor-derived collagen-binding (matrix-targeting) motifexpressed from the CMV promoter, and a retroviral vector bearing wereplaced on separate plasmids, each containing the SV40 origin ofreplication. The resulting vectors are referred to as Mx-GM-CSF,CAE-GM-CSF, Mx null, and Mx-nBg to indicate the envelope and geneencoded in each vector. Mx-GM-CSF is a matrix (i.e. collagen)-targetedvector bearing a human GM-CSF construct. CAE-GM-CSF, is a non-targetedvector bearing the wild type MLV 4070A env protein (Morgan et al.,1993). Mx-null is a matrix-targeted vector bearing only the neo gene,and Mx-nBg, is a collagen-matrix-targeted vector bearing a nucleartargeted β-galactosidase gene. The collagen-matrix -targeting resultsfrom the insertion of a collagen-binding peptide derived from human vonWillebrand factor into the MLV 4070A env protein (Hall et al., 2000).Viral titers in murine NIH3T3 cells were determined as previouslydescribed, based on expression of the β galactosidase or neomycinphosphotransf erase resistance, neo gene (Skotzko et al., 1995). Viraltiter was expressed as number of nuclear β-galactosidase expressingcolonies or G418 resistant colony forming units (cfu)/ml, and rangedfrom 0.3−1.8×10⁷ cfU/ml.

Example 3 GM-CSF Production in Transduced NIH3T3 and 293T Cell Cultures

[0048] To assess the production and secretion of GM-CSF,immunohistochemical staining of transduced cells was conducted using apolyclonal goat antibody raised against a peptide, N1 9, mapping at theamino terminus of human GM-CSF (Santa Cruz Biotechnology, Inc.). GM-CSFproduction was measured in culture medium collected over 3 days inMx-GM-CSF transduced NIH3T3 and transfected 293T producer cell culturesusing commercially available ELISA kits supplied by R&D Systems, Inc.

[0049] Immunoreactive human GM-CSF was noted in 40-50% of transducedNIH3T3 cells and 70-80% of transfected 293T cells (n=4 each group),while GM-CSF production was 32 ng/ml in transduced NIH3T3 cell culturesand 100 ng/ml in transfected 293T cell cultures (FIG. 2).

Example 4 In Vivo Gene Transfer Studies

[0050] In vivo gene transfer studies were conducted in compliance with aprotocol approved by the University of Southern California InstitutionAnimal Care and Use Committee. To evaluate the efficiency of targetedgene delivery based on the enforced expression of nuclearβ-galactosidase and GM-CSF transgenes in vivo, subcutaneous tumorxenografts were established in 8 week old ˜25 gm athymic nu/nu mice bysubcutaneous implantation of 1×10⁷ MiaPaca2 cells. When the tumors havereached a size of ˜20 mm³, 200 μl of either Mx-nBg marker vector,Mx-GM-CSF vector, a non-targeted CAE-GM-CSF vector, Mx-null or phosphatebuffered saline (PBS, pH 7.4), was injected directly into the tail veindaily for 10 days (cumulative vector dose: 2×10⁷ cfu for each vector).The mice were sacrificed by cervical dislocation one day aftercompletion of one treatment cycle.

[0051] Histologic examination of harvested tumor nodules andimmunohistochemical analysis for the presence of β-galactosidasetransgene (Gordon et al., 2000b).

[0052] Harvested tumor nodules were either quick frozen in liquidnitrogen or fixed in 10% formalin. Formalin-fixed tissue sections werestained with hematoxylin-eosin. Transduction efficiency was determinedby immunohistochemical staining of the tumor nodules, using a mousemonoclonal antibody directed against the β-galactosidase antigen(GAL-40, Sigma, St. Louis Mo., USA) followed by analysis using anOptimas imaging system (Optimas Corporation, Bothell, Wash., USA).Transduction efficiency (expressed as %) is determined by counting thenumber of β-galactosidase positive cells in three high power fields pertumor nodule, divided by the total number of cells×100.

[0053] Immunostaining for GM-CSF protein in tumor tissues (Miller etal., 2000).

[0054] For detection of the human GM-CSF protein in harvested tumors,tumor tissues harvested at the end of the experiment were frozenimmediately in liquid nitrogen and kept at −70° C. until used. Five μmtissue sections were cut and fixed in ice-cold acetone for 10 min. Agoat polyclonal anti-GM-CSF monoclonal antibody was supplied by SantaCruz Biotechnology, Inc. The slides were blocked with 2% normal goatserum for 10 min, washed in phophate-buffered saline, and the primaryantibody to GM-CSF diluted at 1:50 was added on the slides for 60 min.Then, the slides were washed three times with PBS and a secondaryantibody, anti-goat IgG conjugated with peroxidase (1:100: VectorLaboratories, Burlingame Calif., USA) was added to the slides for 30min. The slides were washed five times with PBS and developed with a DABsubstrate kit (Vector Laboratories). After counterstain with methylgreen, the slides were examined for presence of brownish-redimmunostaining material indicating presence of the GM-CSF transgene intumor tissues. The efficiency of gene delivery (expressed as %) isdetermined by counting the number of immunoreactive cells to theanti-GM-CSF antibody in three high power fields per tumor nodule,divided by the total number of cells x 100.

[0055] Immunoreactive β-galactosidase was noted in ˜35% of cellsthroughout the tumor nodules of Mx-nBg vector-treated animals (FIG. 3B),while no immunoreactive protein was noted in the control Mx-nullvector-treated tumors (FIG. 3A). Consistent with the high leveltransduction of tumor nodules noted previously with the Mx-nBg vector,immunreactive human GM-CSF protein was noted in ˜35% of cells throughoutthe tumor nodules of Mx-GM-CSF vector-treated mice (FIG. 4B-C) comparedto <1% in CAE-GM-CSF vector-treated and Mx-null vector-treated mice(FIG. 4A).

Example 5 Recruitment of Host Mononuclear Cells into the Tumor Nodulesof Mice Treated with the Mx-GM-CSF Retroviral Vector

[0056] Extensive infiltration of host mononuclear cells was noted intumor nodules of Mx-GM-CSF-treated mice (FIGS. 5B&D) compared to minimalmononuclear infiltration in CAE-GM-CSF, Mx-null vector- and PBS-treatedanimals (FIGS. 5A&C). Within the tumor nodule, the tumor infiltratinglymphocyte (TIL) to tumor cell (T) ratio was 1:20 in Mx-GM-CSF-treatedmice compared to 1:90 in nontargeted CAE-GM-CSF vector-treated mice, and1:99 in Mx-null and PBS-treated animals. These findings indicatesuccessful recruitment of host mononuclear cells into the tumor noduleby GM-CSF secreting cells in Mx-GM-CSF vector-treated mice.

Example 6 2Bimodal Therapy in Vivo Efficacy Studies

[0057] To evaluate bimodal therapy a matrix a targeted retroviral vectorcarrying a cytocidal gene, specifically a dominant negative mutantcyclin G1 gene was constructed using the methods described herein above.The resulting vector is desiganted Mx-dnG1. These studies were conductedin immune competent Balb/c mice (weighing ˜25 gms), in compliance with aprotocol approved by the University of Southern California InstitutionAnimal Care and Use Committee.

[0058] To evaluate the efficacy of single versus dual gene therapy invivo, subcutaneous tumors were established in immune competent Balb/cmice by subcutaneous implantation of 1×10⁶ murine colon-26 cancer cells(NCI, Bethesda Md., USA). Three days later, 200 ml of either the Mx-dnG1vector (cumulative dose: 1.7×10⁸ cfu/mouse), Mx-GM-CSF vector(cumulative dose: 1.7×10⁸ cfu/mouse), a combination of Mx-dnG1 andMx-GM-CSF vectors (cumulative dose of each vector: 8×10⁷ cfu/ml) or anequivalent volume of phosphate buffered saline (PBS, pH 7.4) placebocontrol was injected intravenously twice a day for 5 days. The size ofthe tumor was measured every 4 days with a Vernier caliper, using theformula for calculating the volume of ellipsoid objects: Tumor Volume,mm³={fraction (4/3)} p r₁ r₂ r₃. At the end of 5 days, the tumors wereresected, and the animals were given a tumor re-challenge one week aftercompletion of vector treatment. The significance of differences in meantumor volumes among the four treatment groups were evaluated usingANOVA.

[0059] To evaluate a dose-dependent response to the Mx-GM-CSF vector invivo, 5×10⁶ cells were implanted subcutaneously, and when the tumorsreached a size of ˜35-40 mm³, 200 ml of either a low-dose (cumulativedose: 8×10⁷ cfu/mouse) or high-dose Mx-GM-CSF vector (cumulative dose:1.7×10⁸ cfu/mouse), or an equivalent volume of phosphate buffered saline(PBS, pH 7.4) placebo control was injected directly into the tail veineach day for 16 days. The mice were sacrificed by cervical dislocationone day after completion of treatment. The significance of differencesin tumor volumes among the three treatment groups was evaluated usingANOVA.

[0060] The combination of the vectors Mx-GM-CSF and Mx-dnG1 induced thegreatest inhibition of tumor growth (p<0.05), and to a lesser extent theMX-dnG1 or Mx-GM-CSF alone. Notably, only half the dose of each vectorwas given to attain the desired anti-tumor effect.

[0061] The combination of immunotherapy with radical surgery,chemotherapy and/or radiation therapy has the potential of eradicatingminimal residual disease. In the United States, over 150 approved Phase{fraction (1/11)} gene therapy protocols involve the use of geneticallyengineered syngeneic or allogeneic cells (tumor cells, T cells,dendritic cells and fibroblasts) for vaccine therapy. Cytokine genesused for immunotherapy include IL-1, TNF, IL-2, IFN-y, IL-4, IL-7 andGM-CSF(Foreman et al., 1993; Fearon et al., 1990; Blankenstein et al.,1991; Asher et al., 1991; Tepper et al., 1994; Dranoffet al., 1993;Knobloch et al, 1991; Gansbacher et al., 1991). Other immunostimulatorymolecules such as the T-cell co-stimulatory molecule B7.1 (Guinan etal., 1994) or a foreign MHC molecule (Nabel et al., 1993) have also beenused to generate anti-tumor immunity.

[0062] The major goal of the use of immunostimulatory cytokines is theactivation of tumor-specific T lymphocytes capable of rejecting tumorcells from patients with low tumor burden or to protect patients fromrecurrence of the disease. Treatment of rodents with cancer xenograftswith this strategy have resulted in regression of pre-existing tumorsand cure of the animals from their disease. Further, some animals haveretained immunological memory and resisted a second challenge with theparental tumor cells (Gilboa, 1996; Mackensen et al., 1997).

[0063] The mechanisms by which transduced cytokines enhance tumorimmunity are the subject of much investigation. Without being bound bytheory, they may act by increasing the recruitment of cytotoxic T cellsable to recognize tumor-specific antigens (e.g. IL-4)(Golumbek et al.,1991) or of CD4-positive cells (e.g. TNF and IL-7) (Asher et al., 1991;Hock et al., 1991), or they may increase tumor antigen expression byupregulating MHC Class I antigens (e.g. IFN-γ) (Watanabe et al., 1989),thereby rendering non-transduced target cells more vulnerable tocytotoxic attack. Alternatively, a combination of these effects may beproduced. For example, IL-2can recruit both cytotoxic T and NK cellsdirectly and can also induce release of the secondary cytokine IFN-γ(Main et al., 1985), which augments MHC expression on tumor cells (Cozeet al., 1995; Ucar et al., 1995; Handgretinger R et al., 1989).Additional (non-lymphocytic) effector mechanisms appear to contribute aswell, since the tumor site is often infiltrated with eosinophils andmacrophages. As a final mechanism, some cytokines such as GM-CSF mayenhance the activity and differentiation of antigen presenting cells,which ingest tumor cells and present their antigens either inassociation with Class I or Class II MHC molecules, thereby recruitingCD8+ or CD4+ tumor specific T lymphocytes (Dranoffet al., 1993).

[0064] The inhibition of subcutaneous tumor growth in nude mice byintravenous administration of a matrix-targeted retroviral vectorbearing a cytocidal/cytostatic cyclin G1 construct (Gordon et al.,2000a) was recently reported. These retroviral vectors displayed acollagen-binding motif derived from von Willebrand coagulation factorthat could target extracellular matrix (ECM) exposed during tumorgrowth, angiogenesis, and metastasis. Enhanced vector penetration andtransduction of tumor nodules was demonstrated and correlated withtherapeutic efficacy without associated toxicity. In the present study,we demonstrated high level GM-CSF secretion in NIH3T3 and 293T cellcultures transduced or transfected, respectively, with a matrix-targetedretroviral vector bearing a human GM-CSF construct, as well as highlevel transduction of subcutaneous human cancer xenografts in nude miceby repeated intravenous injections of the Mx-GM-CSF vector. Further, theenforced expression and secretion of GM-CSF by transduced cellsthroughout the tumor nodule resulted in recruitment of host mononuclearcells into the tumor nodule. These studies were conducted in athymicnude mice lacking cytotoxic T cells. In immune competent mice with acomplete repertoire of T lymphocytes and antigen-presenting B cells,dendritic cells and macrophages, regression of the tumor nodules andestablishment of long term antitumor immunity would be expected tooccur, particularly when tumor antigens are presented simultaneously byconcomitant administration of a matrix-targeted vector bearing acytocidal gene.

[0065] The data demonstrates that a targeted retroviral vectorcomprising a cytokine gene, such as a matrix (i.e. collagen)-targetedretroviral vector bearing a human GM-CSF construct can be injectedintravenously to achieve (i) high level efficiency of cytokine genedelivery into solid tumors, (ii) secretion of GM-CSF by tumor cellswithin the solid tumor, and (iii) recruitment of host mononuclear cells(tumor infiltrating lymphocytes, TIL) into the GMCSF secreting tumornodules for eradication of primary, metastatic, inaccessible or minimalresidual disease.

[0066] It is to be understood that while the invention has beendescribed in conjunction with the above embodiments, that the foregoingdescription and the following examples are intended to illustrate andnot limit the scope of the invention. Other aspects, advantages andmodifications within the scope of the invention will be apparent tothose skilled in the art to which the invention pertains.

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What is claimed is:
 1. A targeted retroviral vector particle comprisinga modified viral surface protein for targeting the vector and a cytokinegene.
 2. The targeted retroviral particle of claim 1, wherein themodified viral surface protein is targeting the extracellular matrix ortumor vasculature.
 3. The targeted retroviral particle of claim 1,wherein the modified viral surface protein is targeting theextracellular matrix.
 4. The targeted retroviral particle of claim 3,wherein the modified viral surface protein is targeting a collagenbinding motif.
 5. The targeted retroviral particle of claim 4, whereinthe modified viral surface protein is targeting the Von Willebrandcoagulation factor.
 6. The targeted retroviral particle of claim 3,wherein the modified viral surface protein is targeting tumorvasculature.
 7. The targeted retroviral particle of claim 1, wherein thecytokine gene is selected from the group consisting of IL-1, TNF, IL-2,IFN-γ, IL-4, IL-7 and GM-CSF.
 8. The targeted retroviral particle ofclaim 7, wherein the cytokine gene is GM-CSF.
 9. A pharmaceuticalcomposition comprising the targeted retroviral vector of claim
 1. 10. Amethod for inhibiting cancer in a subject comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 9. 11. The pharmaceutical composition of claim 9, furthercomprising a targeted retroviral vector particle comprising a modifiedviral surface protein for targeting the vector and a cytocidal gene. 12.The pharmaceutical composition of claim 11, wherein targeted retroviralparticle comprising a modified viral surface protein for targeting thevector and a cytocidal gene is targeting the extracellular matrix ortumor vasculature.
 13. The pharmaceutical composition of claim 11,wherein the cytocidal gene is selected from the group consisting oftumor suppressor genes, thymidine kinases or mutated cyclin genes. 14.The pharmaceutical composition of claim 13, wherein the mutated cyclingene is a dominant negative mutation of a cyclin G1 gene.
 15. A methodfor inhibiting cancer in a subject comprising administering to thesubject an effective amount of the pharmaceutical composition of claim11.
 16. A composition comprising a targeted retroviral vector particlecomprising a modified viral surface protein for targeting the vector tothe Von Willebrand coagulation factor and a cytokine gene and a targetedretroviral vector particle comprising a modified viral surface proteinfor targeting the vector to the Von Willebrand coagulation factor and acytocidal gene.
 17. The composition of claim 16, wherein the cytocidalgene is a mutated cyclin gene.
 18. The composition of claim 17, whereinthe cytocidal gene is a dominant negative mutation of the cyclin G1gene.
 19. The composition of claim 16, wherein the cytokine is selectedfrom the group consisting of IL-1, TNF, IL-2, IFN-γ, IL-4, IL-7 andGM-CSF.
 20. The composition of claim 19, wherein the cytokine is GM-CSF.21. The composition of claim 16, further comprising a pharmaceuticalexcipient.
 22. A method for inhibiting cancer in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim 21.